Chaperone machines for protein folding, unfolding and disaggregation.

Department of Crystallography, Institute for Structural and Molecular Biology, Birkbeck College London, UK.

Abstract

Molecular chaperones are diverse families of multidomain proteins that have evolved to assist nascent proteins to reach their native fold, protect subunits from heat shock during the assembly of complexes, prevent protein aggregation or mediate targeted unfolding and disassembly. Their increased expression in response to stress is a key factor in the health of the cell and longevity of an organism. Unlike enzymes with their precise and finely tuned active sites, chaperones are heavy-duty molecular machines that operate on a wide range of substrates. The structural basis of their mechanism of action is being unravelled (in particular for the heat shock proteins HSP60, HSP70, HSP90 and HSP100) and typically involves massive displacements of 20-30 kDa domains over distances of 20-50 Å and rotations of up to 100°.

a ∣ In the ADP-bound or nucleotide-free state, the nucleotide-binding domain (green; Protein Data Bank (PDB) code: 3HSC) of heat shock protein 70 (HSP70) is connected by a flexible linker to the substrate-binding domain (blue; PDB code: 1DKZ), with the lid domain (red) locking a peptide substrate (yellow) into the binding pocket. A side view of the substrate domain is shown on the right. A cartoon depicting the two-domain complex is shown below. The bound nucleotide is shown in space filling format. b ∣ In the ATP-bound state, the lid opens, and both the lid and the substrate-binding domain dock to the nucleotide-binding domain (PDB code: 4B9Q). The corresponding cartoon of this conformation is shown below. When ATP binds, the cleft closes, triggering a change on the outside of the nucleotide-binding domain that creates a binding site for the linker region. Linker binding causes the substrate-binding domain and the lid domain to bind different sites on the nucleotide-binding domain, resulting in a widely opened substrate-binding site that enables rapid exchange of polypeptide substrates. After hydrolysis, the domains separate and the lid closes over the bound substrate. Such binding and release of extended regions of polypeptide chain are thought to unfold and stabilize non-native proteins either for correct folding or degradation.

Crystal structures of heat shock protein 90 (HSP90) dimers in an open, unliganded state (Protein Data Bank (PDB) code: 2IOQ) (part a), a partly closed, ADP-bound state (PDB code: 2O1V) (part b) and in a closed, ATP-bound state (PDB code: 2CG9) (part c), are shown, and the amino-terminal domain (green), the middle domain (yellow) and the carboxy-terminal domain (blue) are indcated. The open form shown is Escherichia coli HptG, the partly closed ADP-bound form is the canine endoplasmic reticulum-associated HSP90 homologue GRP94 and the ATP-bound form (shown is the ATP analogue AMP-PNP) is yeast Hsc82 (heat shock cognate 82). Nucleotides are shown in space filling format. ATP favours binding to the closed form (part c), whereas hydrolysis or nucleotide release is favoured by a range of more open states (parts a,b). Opening and closing of the cleft are thought to mediate the action of HSP90 on its substrates, although the mechanisms underlying HSP90 action remain largely unclear. The electron microscopy map of HSP90 in complex with the cofactor p50 and its substrate cyclin-dependent kinase 4 (CDK4) is shown (part d). Extra density of the side of this asymmetric complex is attributed to the cofactor and substrate.

a ∣ Overview of unliganded (apo) GroEL (Protein Data Bank (PDB) code:1OEL) (left) and the GroEL–GroES complex (PDB code: 1SVT) (right). The overall shapes are shown as blue surfaces, with three subunits coloured by domain in red, green and yellow in apo GroEL. One subunit of GroEL and one of GroES (cyan) are highlighted in the GroEL–GroES complex. b ∣ Conformation of a GroEL subunit in the apo form (left) and the GroES-bound form (right), with GroEL key sites indicated (GroES is not shown). c ∣ Cartoons of complexes with folding proteins. Hydrophobic surfaces and residues are shown in yellow and polar residues in green. d ∣ Cut open view of the cryo-electron microscopy structure (Electron Microscopy Data Bank code: EMD-1548) of GroEL (PDB code: 1AON) in complex with bacteriophage 56 kDa capsid protein (gp31) (PDB code: 1G31), with a non-native gp23 (PDB code: 1YUE) bound to both rings. The pink density in the folding chamber corresponds to newly folded gp23, and the yellow density in the open ring is part of a non-native gp23 subunit. The corresponding atomic structures are shown embedded in the electron microscopy density map, except for the non-native substrate, which is unknown and only partially visualized owing to disorder. The open ring with its hydrophobic lining is the acceptor state for non-native polypeptides, and binding to multiple sites may facilitate unfolding. ATP and GroES binding to the chaperonin create a protected chamber with a hydrophilic lining that allows the encapsulated protein to fold.

a ∣ The two types of heat shock protein 100 (HSP100) sequences are shown schematically, with either a single or two tandem AAA+ domains. The characteristic Walker A and B sites are shown in red. b ∣ The HslUV ATPase–protease complex is shown as a cartoon on the left, and the atomic structure is shown on the right (Protein Data Bank (PDB) code: 1G3I). c ∣ Top view of the asymmetric ClpX crystal structure (PDB code: 3HWS). The four bound ADP molecules are shown in space-filling format and Tyr side chains on the pore loops are shown as magenta sticks. d ∣ Side view section of ClpX showing the pore with three of the Tyr sites at different heights.

The crystal structure of a ClpB subunit (Protein Data Bank (PDB) code: 1QVR) (part a) and a schematic representation of the three-tiered hexamer are shown, with one ClpB coiled-coil domain (dark blue) bound to heat shock protein 70 (HSP70; with the nucleotide-binding domain shown in green and the substrate-binding domain in blue (PDB code: 4B9Q)) (part b). The ClpB–Hsp70 complex is derived from the model in combined with the structure of domain-docked HSP70 from . The motif 2 sequence in the coiled-coil domain is highlighted in pink. A substrate polypeptide (yellow) is being extracted from an aggregate and threaded through the ClpB channel.